Many Gram-negative pathogens utilize the PhoP-PhoQ regulatory system to sense changes in levels of divalent cations and modulate the expression of virulence genes that play a critical role in enhancing infection processes. Our research focuses on the PhoP-PhoQ system found in the mutualistic tsetse fly endosymbiont, Sodalis glossinidius, which controls the expression of a number of essential symbiosis determinants in vivo. While the basic functions and regulatory targets of PhoP-PhoQ appear to be well conserved between pathogens and S. glossinidius, our preliminary studies show that the S. glossinidius PhoQ protein has lost the ability to respond to signals (magnesium, antimicrobial peptides and acidic pH) that play a crucial role in PhoP-PhoQ-mediated gene regulation in opportunistic host-associated bacteria. The changes observed in the functionality of this regulatory system are consistent with a switch in lifestyle from opportunism to obligate host association, in which PhoP-regulated genes are required to be constitutively expressed. To date, very few studies have focused on molecular interactions between insect hosts and their mutualistic bacterial symbionts. S. glossinidius provides an excellent model for study because it is one of only a small number of mutualistic insect symbionts that have been cultivated under axenic conditions in the laboratory. In addition, a whole genome sequence is available for S. glossinidius, and our laboratory has recently developed robust techniques facilitating the genetic manipulation of this symbiont. We have also recently identified a non insect-associated progenitor of the Sodalis-allied clade of insect endosymbionts, and this will enable us to determine the ancestral functions of PhoP-PhoQ. Thus, in the current project we will identify all genes that are regulated by PhoP-PhoQ in S. glossinidius and its extant progenitor using census RNA sequencing (RNA-Seq) methods. We will then generate S. glossinidius mutants that are either (i) modulated in their ability to sense environmental signals via PhoQ, or (ii) lac genes encoding critical symbiosis determinants that are regulated by PhoP-PhoQ. These mutants will then be assessed for their ability to initiate and maintain symbiotic interactions in tsetse flies. The primary goal of the proposed research is to understand how the functions of PhoP-PhoQ have been modulated in the transition to obligate insect association. The secondary goal is to further the development of techniques that will allow S. glossinidius to be used as a platform for the expression of anti-trypanosomal peptides in a novel control strategy, aimed at reducing the vectorial capabilities of tsetse flies, which are important vectors of African trypanosomiasis, causing sleeping sickness in man and nagana in domestic livestock. PUBLIC HEALTH RELEVANCE: The proposed research focuses on understanding the evolution and function of a two-component regulatory system (PhoP-PhoQ) that plays an important role in modulating the virulence of bacterial pathogens. This project will enable us to understand how obligate, mutualistic, host-associated bacteria utilize PhoP-PhoQ to overcome attack from the host immune system and maintain symbiotic interactions with their insect host. In addition, this research will facilitate the development of techniques that will allow the tsetsefly symbiont, Sodalis glossinidius, to be used as a platform for the expression of anti-trypanosomal peptides in a paratransgenic disease control strategy that represents a novel approach for the control of the African trypanosomiasis.